The study was conducted to detect potential adverse effects of the microbicidal composition according to the present invention by measuring susceptibility of mice to infection with herpes simplex virus type 2 (HSV-2), the virus that most commonly causes genital herpes.
The mouse HSV-2 vaginal transmission model is used by Richard Cone at Johns Hopkins University, Baltimore, USA, to assess toxicities associated with microbicides that could lead to susceptibility to pathogens such as HSV-2.
Methods
Mouse Model:
Prior to the susceptibility assessment, female CF-1 mice (Harlan, Indianapolis, Ind., USA) 6-8 weeks old are progestin treated (Depo Provera®, medroxyprogesterone acetate) to increase HSV-2 susceptibility, and to make the mice more uniform in terms of susceptibility than mice at different stages of the oestrous cycle.
Viral Inoculum:
Strain G of HSV-2, 5×108 TCID50/mL.
Procedures:
20 μL of the microbicidal composition according to the present invention was administered to the vagina followed 12 hours later by administration of a low-dose inoculum of HSV-2 (0.1 ID50) delivered in 10 μL of Bartels medium. Control animals received 20 μL of PBS instead of test product.
The inoculum is delivered 12 hours after application of the test product because previous experiments showed that this was the time at which peak susceptibility to HSV-2 infection occurred following administration of nonoxynol-9.
In this study, a total of 40 mice received the microbicidal composition according to the present invention and a total of 40 mice received PBS.
Results
Only 1 out of the 40 mice treated with the microbicidal composition according to the present invention became infected with HSV-2. In contrast, 7 out of 40 mice in the control group became infected. In other words, there was no increase in susceptibility following administration of the microbicidal composition according to the present invention.
In previous studies, 29 out of 42 animals treated with nonoxynol-9, 20 out of 30 animals treated with microbicide ingredient 1, and 25 out of 41 animals treated with microbicide ingredient 2, became infected.
To determine relative susceptibility of the mice in previous studies, two groups of control mice were treated with PBS for every group of mice treated with test product. One control group was inoculated with 0.1 ID50, while the other was inoculated with 10 ID50. The fraction of animals infected in each control group was then used to construct a dose-response graph (fraction infected vs. log ID), drawing a linear interpolation between the low and high dose points. The fraction of mice infected in the test group was then plotted on this graph to determine the effective ID of the low-dose inoculum in this test group. Relative susceptibility was defined as the effective ID the low-dose inoculum delivered to the test mice divided by the ID it delivered to the control animals.
Animals treated with nonoxynol-9 were 29.7 times more susceptible to HSV-2 infection than the control animals (P<0.001, Fishers exact two-sided t-test), while animals treated with microbicide ingredients 1 and 2 were 29.1 (P<0.001) and 17.5 (P<0.001) times more susceptible, respectively.
Conclusion
The microbicidal composition according to the present invention does not appear to lead to increased susceptibility in the mouse-model of HSV-2 infection. Nonoxynol-9 and other detergent microbicides may lead to increased susceptibility.
Reference
    Cone R A, Hoen T E, Wang X X & Moench T R. Microbicidal Detergents Increase HSV Susceptibility in Mice Without Causing Visible Epithelial Defects. Abstract # 02421, “Microbicides 2004” Conference, London, UK; March 2004.
It will be appreciated that variations and modifications may be made to the invention as broadly described herein, other than those specifically described without departing from the spirit and scope of the invention. It is to be understood that this invention extends to include all such variations and modifications.